Nonapeptide and decapeptide analogs of LHRH useful as LHRH antagonists

ABSTRACT

Synthetic nona- and decapeptide LHRH antagonist analogs are disclosed, having a sterically hindered guanidino-substituted arginyl or homoarginyl residue at position 8, with no arginyl substituent at position 6.

BACKGROUND OF THE INVENTION

Luteinizing hormone (LH) and follicle stimulating hormone (FSH) arereleased from the anterior pituitary gland under the control ofluteinizing releasing hormone (LHRH) produced in the hypothalamicregion. LH and FSH act on the gonads to stimulate the synthesis ofsteroid hormones and to stimulate gamete maturation. The pulsatilerelease of LHRH, and thereby the release of LH and FSH, controls thereproductive cycle in domestic animals and humans.

LHRH also affects the placenta, and therefore the gonads indirectly, bycausing the synthesis and release of chorionic gonadotropin (CG).

Antagonists of LHRH are useful for the control of fertility. Suchantagonists block ovulation in the female and suppress spermatogenesisin the male. Related to these effects is a suppression of normalcirculating levels of sexual steroids of gonadal origin, causingreduction in accessory organ weight in the male and the female. Indomestic animals this effect suppresses sexual cycling and behavior(promoting weight gain in a feed-lot situation), induces abortion inpregnant animals, and in general, acts as a chemical sterilant.

The natural releasing hormone LHRH is a decapeptide comprised ofnaturally occuring amino acids (which have the L-configuration exceptfor the achiral amino acid glycine). Its sequence is as follows:##STR1## Analogs of this natural material are often described inabbreviated form by showing the nature of the substituent of a givenamino acid, superscribed by its location, followed by "LHRH." Manyanalogs of LHRH have been studied, and a very large majority are ofinsufficient biological activity to be clinically useful. However,certain select modifications have a potentiating effect on agonistbiological activity. A significant enhancement to agonist activity isobtained by changing the 6-position residue from Gly to a D-amino acid.

In addition to agonists, analogs have been prepared which arecompetitive antagonists to LHRH, all of which require deletion orreplacement of the histidyl residue at position 2: Vale, W., et al.,Science, 176: 933 (1972). In general, it appears that a D-amino acidplaced in the sequence at that position gives the best activity: Rees,R. W. A., et al., J. Med. Chem. 17: 1016 (1974).

Moreover, adding a modification at the 6 position (which, without themodification at position 2, results in the agonist activity cited above)enhances the antagonist activity of the 2-modified analogs: Beattie, C.W., et al, J. Med. Chem., 18: 1247 (1975); Rivier, J., et al., Peptides1976 p. 427, Editions de l'Universite de Bruxelles, Belgium (1976).

Against the background of these two major alterations, which result inmore potent LHRH antagonists, additional increments in antagonistactivity may be had by modifying positions 1, 3, 5 and/or 10 in thealready 2, 6 modified peptide. Coy, D. H., et al Peptides 1976, p. 462,Editions de l'Universite de Bruxelles, Belgium (1976); Rivier, J. E., etal, Life Sci. 23: 869 (1978); Dutta, A. S., et al, Biochem Biophys. Res.Commun. 81: 382 (1978), Humphries, J., et al, Biochem. Biophys. Res.Commun., 85: 709 (1978). It has also been shown that N-acylation of theamino acid at position 1 is helpful; Channabasavaia, K., et al, Biochem.Biophys. Res. Commun. 81: 382 (1978); Coy, D. H., et al,Peptides.--Structure and Biological Function p. 775, Pierce Chemical Co.(1979). Additionally, a highly potent antagonist containing a D-Arg⁶substitution, (N-Ac-D-pCl-Phe¹, D-pCl-Phe², D-Trp³, D-Arg⁶, D-Ala¹⁰)LHRH, has been published by D. H. Coy, Endocrinology, 110, 1445 (1982).

Unfortunately, although the class of LHRH analogs containing a D-Arg⁶substitution were found to be potent antiovulatory substances, they werealso potent mast cell degranulating substances, Schmidt et al.,Contraception, 29, 283 (1984), and caused edema in vivo. Thus, forexample, (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Trp³, D-Arg⁶)LHRH has an ED₅₀=0.2 μg/ml for histamine release from rat mast cells in vitro. This sidereaction is of clinical importance because of the potential lifethreatening nature of the ensuing anaphylactoid reaction.

It is well known in the art that molecules containing positivecharge(s), especially multiple positive charges, in association withhydrophobicity are potent mast cell degranulators: Foreman and Jordan,Agents and Actions, 13, 105 (1983). An initial attempt to circumventthis problem in analogs containing two Arg residues (i.e., in positions6 and 8) was to increase the space between the residues (e.g.,(N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Trp³, Arg⁵, D-Tyr⁶, D-Ala¹⁰)LHRH, (ED₅₀=2 μg/ml for histamine release, R. Roeske, personal communication).While this led to a decrease in the potency of the analog fordegranulation of mast cells and release of histamine, the analog stillhad many fold greater anaphylactoid potency than LHRH, for which ED₅₀ is328 μg/ml for histamine release.

R. Roeske (personal communication) has incorporated Lys(iPr) intopositions 6 and 8 in conjunction with a D-pCl-Phe residue in position 2with retention of high antiovulatory potency and decreased histaminerelease (e.g., (N-Ac-D-Nal(2)¹, pCl-Phe², D-Trp³, D-Lys(iPr)⁸,Lys(iPr)⁶, D-Ala¹⁰)LHRH; ED₅₀ =6.6 μg/ml for histamine release). In oneanalog, hArg(Et₂) was incorporated into position 8 with a similar degreeof histamine release potency (i.e., (N-Ac-D-Nal(2)¹, D-αMe-pCl-Phe²,D-Pal(3)³, D-Arg⁶, hArg(Et₂)⁸, D-Ala¹⁰)LHRH; ED₅₀ =4.9 μg/ml forhistamine release). However, it can be seen that these analogs are stillpotent histamine releasing agents compared to LHRH.

Thus, the presently known set of analogs still has significantpossibility for toxicity and other side effects.

SUMMARY OF THE INVENTION

The present invention refers to novel, highly potent nonapeptide anddecapeptide analogs of LHRH with minimal histamine releasing potency forwhich a replacement at position 8 by a sterically hinderedguanidino-substituted arginyl residue, coupled with avoidance of anarginyl substituent at position 6, is a critical feature. The inventionis also directed to various methods of use of these compounds and topharmaceutical compositions therefor.

More specifically, the present invention relates to compounds of theformula ##STR2## or a pharmaceutically acceptable salt thereof, wherein:

A is an amino acyl residue selected from the group consisting of eitherthe D- or the L-isomer of: N-Ac-D,L-Δ³,4 -prolyl, N-Ac-D,L-prolyl,N-Ac-D,L-phenylalanyl, N-Ac-D,L-p-chlorophenylalanyl,N-Ac-D,L,-p-fluorophenylalanyl, N-Ac-3-(1-naphthyl)-D,L-alanyl,N-Ac-3-(2-naphthyl)-D,L-alanyl, andN-Ac-3-(2,4,6-trimethylphenyl)-D,L-alanyl;

B is an amino acyl residue selected from the group consisting ofD-phenylalanyl, D-p-chlorophenylalanyl, D-p-fluorophenylalanyl,D-p-nitrophenylalanyl, 2,2-diphenylglycol,D-α-methyl-p-chlorophenylalanyl and 3-(2-naphthyl)-D-alanyl;

C is an amino acyl residue selected from the group consisting ofD-tryptophanyl, D-phenylalanyl, 3-(3-pyridyl)-D-alanyl, and3-(2-naphthyl)-D-alanyl;

D is an amino acyl residue selected from the group consisting ofL-phenylalanyl, L-tyrosyl, and 3-(3-pyridyl)-alanyl, arginyl, or G;

E is 3-(2-naphthyl)-D-alanyl, 3-(3-pyridyl)-D-alanyl, D-tyrosyl,D-tryptophanyl, D-nicotinyl-lysyl, pyridylacetyl-lysyl, D-Glu(AA) or G;

F is an amino acyl residue selected from the group consisting ofL-leucyl, L-norleucyl, L-phenylalanyl, L-tryptophanyl, and3-(2-naphthyl)-L-alanyl;

G is an amino acyl residue selected from the group consisting of theradicals represented by the following structural formulas: ##STR3##wherein n is 1 to 5;

R¹ is alkyl of 1 to 6 carbon atoms or fluoroalkyl;

R² is hydrogen or R¹ ; or R¹ --HN--C═NR² is a ring represented by thefollowing structural formulas: ##STR4## wherein m is 1 to 4; A ishydrogen or alkyl of 1 to 6 carbon atoms; and X is halo or A; and##STR5## wherein R³ is hydrogen, alkyl of 1 to 6 carbon atoms, phenyl orphenylloweralkyl; and

J is D-alaninamide; D-leucinamide; glycinamide; or --NHR⁴ wherein R⁴ islower alkyl or NHCONH₂.

Another aspect of the invention relates to a method of treatingendometriosis in a female mammalian subject, which method comprisesadministering to said subject an effective amount of a compound ofFormula I or a pharmaceutical composition containing an effective amountof a compound of Formula I.

Still another aspect of the invention relates to a method of treatingprostatic hypertrophy in a male mammalian subject, which methodcomprises administering to said subject an effective amount of thecompound of Formula I or a pharmaceutical composition containing aneffective amount of the compound of Formula I.

Yet another aspect of the invention relates to a method of treatingprecocious puberty in a human subject, which method comprisesadministering to said subject an effective amount of the compound ofFormula I or a pharmaceutical composition containing an effective amountof the compound of Formula I.

A further aspect of the invention relates to a method of interruptingheat in an animal, which method comprises administering to said animalan effective amount of the compound of Formula I or a pharmaceuticalcomposition containing an effective amount of the compound of Formula I.

A still further aspect of the invention relates to a method ofterminating pregnancy in an animal, which method comprises administeringto said animal an effective amount of the compound of Formula I or apharmaceutical composition containing an effective amount of thecompound of Formula I.

An additional aspect of the invention relates to a method of inhibitingspermatogenesis in a male mammalian subject, which method comprisesadministering to said subject an effective amount of the compound ofFormula I or a pharmaceutical composition containing an effective amountof the compound of Formula I.

Another important aspect of the invention relates to a method ofpreventing ovarian hyperstimulation in response to exogenousgonadotropins in a human female, which method comprises administering tosaid subject an effective amount of the compound of Formula I or apharmaceutical composition containing an effective amount of thecompound of Formula I.

An additional important aspect of the invention relates to a method oftreating premenstrual syndrome in a human female, which method comprisesadministering to said subject an effective amount of the compound ofFormula I or a pharmaceutical composition containing an effective amountof the compound of Formula I.

A still additional aspect of the invention relates to a method forpreventing ovulation in a human female, which method comprisesadministering to said subject an effective amount of the compound ofFormula I or a pharmaceutical composition containing an effective amountof the compound of Formula I.

A final aspect of the invention relates to a pharmaceutical compositionfor inhibiting ovulation in a mammalian female subject; preventingovarian hyperstimulation in response to exogenous gonadotropins,treating premenstrual syndrome, or treating endometriosis in a femalehuman subject; for treating prostatic hypertrophy or inhibitingspermatogenesis in a male mammalian subject; or for treating precociouspuberty in a human subject; or interrupting heat in a female animal; orterminating pregnancy in a female mammalian subject; comprising aneffective amount of the compound of Formula I in admixture with at leastone pharmaceutically acceptable excipient.

DETAILED DESCRIPTION OF THE INVENTION

The replacement of the L-histidyl residue which is at position 2 in LHRHwith one of the residues herein specified is a requirement to convertthe peptide to an LHRH antagonist. The replacement of the glycyl residueat position 6 in LHRH with one of the residues specified as E gives adramatic enhancement of the antagonist effect. The substitutionsdisclosed herein at positions 1, 2, 3, 5, 7 and 10 are further helpfulin enhancing the antagonist activity. The substitution G in position 8provides a profound diminution in the histamine releasing potency of theanalogs when the substitution in position 6 is other than Arg, and iscritical for their use as safe drugs.

ABBREVIATIONS AND DEFINITIONS

As set forth above, and for convenience in describing this invention,the conventional abbreviations for the various common amino acids areused as generally accepted in the peptide art as recommended by theIUPAC-IUB Commission on Biochemical Nomenclature, Biochemistry, 11, 1726(1972). All peptide sequences mentioned herein are written according tothe generally accepted convention whereby the N-terminal amino acid ison the left and the C-terminal amino acid is on the right.

The abbreviations herein represent L-amino acids, with the exception ofthe achiral amino acid glycine, and with the further exception of anyunnatural or natural amino acids which are achiral, or are otherwisedesignated as D- or D,L-.

It should be noted that when J=NH--C(O)--NH₂, the C-terminus is anaza-glycinamide residue.

The abbreviation D-Glu(AA) represents the anisole adduct to a D-Gluresidue to form a para-methoxy-phenylketone (at the carboxyl terminus ofthe glutamic acid side chain), i.e., Glu(pMeO-Ph).

Certain other abbreviations will be useful in describing the invention.The present invention employs replacements of amino acids in the naturalLHRH peptide by amino acids which do not occur in nature. Particularlycommonly employed among these are the following:

    ______________________________________                                        Amino acid residue   Abbreviation                                             ______________________________________                                        3-(2-naphthyl)-alanyl                                                                              Nal(2)                                                   3-(p-fluorophenyl)-alanyl                                                                          pF--Phe                                                  3-(p-chlorophenyl)-alanyl                                                                          pCl--Phe                                                 3-(3-pyridyl)-alanyl Pal(3)                                                   N,N' --guanidino-dimethyl-                                                                         Dmh, or hArg(Me).sub.2                                   homoarginyl                                                                   N,N' --guanidino-(diethyl)-                                                                        Deh, or hArg(Et).sub.2                                   homoarginyl                                                                   N,N' --guanidino-(dipropyl)-                                                                       Dph, or hArg(Pr).sub.2                                   homoarginyl                                                                   N,N' --guanidino-(diisopropyl)-                                                                    Dih, or Arg (iPr).sub.2                                  homoarginyl                                                                   N,N' --guanidino-(dihexyl)-                                                                        Dhh, or hArg(hexyl).sub.2                                homoarginyl                                                                   N,N' --guanidino-(ethano)-                                                                         Eha or hArg(CH.sub.2).sub.2                              homoarginyl                                                                   N,N' --guanidino-(propano)-                                                                        Pha, or hArg(CH.sub.2).sub.3                             homoarginyl                                                                   N,N' --guanidino-bis-(2,2,2-                                                                       Bth, or hArg(CH.sub.2 CF.sub.3).sub.2                    trifluoroethyl)-                                                              homoarginyl                                                                   N--guanidino-(ethyl)-                                                                              Meh, or hArg(Et)                                         homoarginyl                                                                   N--guanidino-(propyl)-                                                                             Prh, or hArg(propyl)                                     homoarginyl                                                                   N--guanidino-(isopropyl)-                                                                          Iph, or hArg(iPr)                                        homoarginyl                                                                   N--guanidino-(butyl)-                                                                              Mbh, or hArg(Bu)                                         homoarginyl                                                                   N,N' --guanidino-(dicyclohexyl)-                                                                   Dch, or                                                  homoarginyl          hArg(cyclohexyl).sub.2                                   N--guanidino-(heptyl)-                                                                             Hha, or hArg(heptyl)                                     homoarginyl                                                                   N--guanidino-(ethyl)-arginyl                                                                       Mea, or Arg(Et)                                          N,N' --guanidino-(diisopropyl)-                                                                    Dia, or Arg(iPr).sub.2                                   arginyl                                                                       N,N' --guanidino-(dicyclohexyl)-                                                                   Dca, or                                                  arginyl              Arg(cyclohexyl).sub.2                                    3-(3-piperidyl)-alanyl                                                                             3-Pia                                                    3-(4-piperidyl)-alanyl                                                                             4-Pia                                                    3-((N.sup.ε --methyl)piperid-4-yl)-                                                        Mpa                                                      alanyl                                                                        3-((N.sup.ε --pentyl)piperid-4-yl)-                                                        Ppa                                                      alanyl                                                                        3-((N.sup.ε --benzyl)piperid-4-yl)-                                                        Bpa                                                      alanyl                                                                        N.sup.ε --Nicotinyl-D-lysyl                                                                Lys(Nic)                                                 N.sup.ε --(3-Pyridyl)acetyl-D-lysyl                                                        Lys(pyridylacetyl)                                       3-(2,4,6-trimethylphenyl)alanyl                                                                    Tmp                                                      2,2-diphenylglycyl   Dpg                                                      ______________________________________                                    

As used herein, the term "pharmaceutically acceptable salts" refers tosalts that retain the desired biological activity of the parent compoundand do not impart any undesired toxicological effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonicacids, polygalacturonic acid; (b) base addition salts formed withpolyvalent metal cations such as zinc, calcium, bismuth, barium,magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; orwith an organic cation formed from N,N'-dibenzylethylene-diamine orethylenediamine; or (c) combinations, of (a) and (b), e.g., a zinctannate salt and the like.

The term "lower alkyl" refers to a straight or branched chain saturatedhydrocarbon radical having from 1 to 4 carbon atoms such as, forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl and tert-butyl. "Alkyl of 1 to 6 carbon atoms" encompasses thesame substituents as lower alkyl but in addition includes radicalshaving 5 or 6 carbon atoms such as, for example, n-pentyl, n-hexyl orbranched 5 or 6 carbon membered radicals. "Alkyl of 1 to 12 carbonatoms" comprises a hydrocarbon radical of 1 to 12 carbon atoms includingthe radicals noted above, except that the radical may have up to 12carbon atoms.

Fluoroalkyl refers to lower alkyl substituted with 1 to 5 fluorineatoms, for example CF₃ CH₂ --, CF₃ --, CF₃ CF₂ CH₂ --, and the like.

Halo refers to fluoro, chloro or bromo.

The abbreviation "N-Ac" refers specifically to the N-acetyl protectinggroup, i.e., an acetyl group attached to a terminal amino acid residueon the amine nitrogen, in conformance with generally acceptednomenclature.

PREFERRED EMBODIMENTS

Compounds which are preferred embodiments of the present invention arethose wherein A is N-Ac-D-Nal(2) or N-Ac-D-pCl-Phe; B is D-pF-Phe orD-pCl-Phe; C is D-Trp, D-Nal(2) or Pal(3); D is Pal(3), Tyr, Arg, Deh,Mbh, Bth, or Pha; E is D-Trp, D-Tyr, D-Nal(2), D-Pal(3), D-Deh, D-Mbh,D-Pha or D-Bth; F is Leu or Phe; G is Deh, Bth, Mbh, or Pha; and J isD-AlaNH₂ or GlyNH₂.

More preferred substitution patterns are those

wherein:

A is N-Ac-D-Nal(2);

B is D-pCl-Phe;

C is D-Trp or D-Pal(3);

D is Tyr, Arg, Deh, Mbh, Bth or Pha;

E is D-Trp, D-Pal(3), D-Nal(2), D-Tyr, D-Deh, D-Mbh, D-Bth or D-Pha;

F is Leu;

G is Deh, Mbh, Bth and Pha; and

J is D-AlaNH₂.

There are three preferred subclasses within this more preferred class:

1. When D is Tyr, E can be any one of either (a) the hydrophobicresidues D-Trp, D-Pal(3), D-Nal(2) or D-Tyr, but most preferably D-Trpor D-Pal(3), or (b) the residues D-Deh, D-Mbh, D-Bth or D-Pha;

2. When D is Arg, E is preferably one of the hydrophobic residues listedin 1(a) above, and most preferably D-Tyr;

3. When D is any of Deh, Mbh, Bth or Pha, E is most preferably D-Tyr orD-Pal(3), but D-Nal(2) and D-Trp are also preferred.

In each of the preferred subclasses, G is Deh, Mbh, Bth or Pha.Particularly preferred are Bth and Deh, and most particularly, Bth.

Thus, examples of the more preferred substitution patterns are:

N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-C-Leu-G-Pro-D-AlaNH₂, wherein C isD-Trp or D-Pal(3) and G is Deh, Mbn, Bth or Pha;

N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-E-Leu-G-Pro-D-AlaNH₂, wherein C isD-Trp or D-Pal(3), E is D-Deh, D-Bth, D-Mbh or D-Pha, and G is theL-form of E as defined in this paragraph;

N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Arg-E-Leu-G-Pro-D-AlaNH₂, wherein C isD-Trp or D-Pal(3), E is D-Trp, D-Pal(3), D-Nal(2), or D-Tyr, and G isDeh, Mbh, Bth or Pha; and

N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Leu-G-Pro-D-AlaNH₂, wherein C is D-Trpor D-Pal(3), D and G are independently Deh, Bth, Mbh or Pha, and E isD-Tyr, D-Nal(2), D-Trp or D-Pal(3).

Additional preferred embodiments are those wherein:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-E-Leu-G-Pro-D-AlaNH₂, wherein E isD-Trp, D-Tyr, or D-Nal(2) and G is Deh, Bth, Mbh, or Pha;

N-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Leu-G-Pro-D-AlaNH₂, wherein D and Gare independently Deh, Bth, Mbh or Pha, and both C and E areindependently D-Trp or D-Pal(3); and

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-G-Pro-D-AlaNH₂,wherein G is Deh, Bth, Mbh, or Pha.

Exemplary preferred embodiments are:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlanNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Deh-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pha-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Nal(2)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal(2)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Nal(2)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-PHa-D-Nal(2)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Deh-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Pha-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal-(3)-Leu-Mbh-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Try-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ; and

N-Ac-D-Nal(2)-D-pF-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH₂.

The scope of the instant invention also includes peptides that may notnecessarily fall within the aforementioned preferred classes, such as:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Nal(2)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-αMe,pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂;

N-Ac-D-Nal(2)-D-αMe,pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂;

N-Ac-D-Nal(2)-D-αMe,pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Glu(AA)-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Glu(AA)-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Glu(AA)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-pCl-Phe-D-Phe-D-Phe-Ser-Phe-D-Lys(Nic)-Nle-Bth-Pro-GlyNH₂ ;

N-Ac-Δ³,4Pro-D-Nal(2)-D-Pal(3)-Ser-Pal(3)-D-Lys(pyridylacetyl)-Phe-Mpa-D-AlaNH.sub.2;

N-Ac-Pro-D-pNO₂ -Phe-D-Trp-Ser-Phe-D-Lys(Nic)-Leu-Ppa-Pro-D-LeuNH₂ ;

N-Ac-D-pF-Phe-D-pF-Phe-D-Trp-Ser-Tyr-D-Tyr-Trp-Bth-Pro-AzaGlyNH₂ ;

N-Ac-D-Nal(1)-Dpg-D-Pal(3)-Ser-Tyr-D-Pal(3)-Nal(2)-Bth-Pro-D-AlaNH₂ ;and

N-Ac-D-Tmp-D-pF-Phe-D-Pal(3)-Ser-Tyr-D-Lys(Nic)-Nal(2)-Bth-Pro-NHEt.

It is generally preferred that the A, B, C, E and J amino acid residuesbe in the form of the D-isomer; and that the D, F and G residues be inthe form of the L-isomer. This stereochemistry is to be understood to berepresented where not otherwise specified.

In all of the above embodiments, the compound may also be prepared as acorresponding pharmaceutically acceptable salt.

Assay Procedures

The compounds of this invention and, particularly, the salts thereof,exhibit surprisingly potent and long lasting LHRH antagonist activity.

A primary measure of LHRH antagonist potency is the ability to inhibitovulation in rats, as assayed by the procedure of Corbin, A. AndBeattie, C. W., Endocrine Res. Commun., 2:1 (1975).

The ability to cause histamine release from rat peritoneal mast cells invitro may be assessed as per Sydbom and Terenius, Agents and Actions,16, 269 (1985) or Siraganian, et al., Manual of Clinical Immunology, 2dEd, N. E. Rose and M. Friedman, Eds., Amer. Soc. Microbiol., Washington,D.C., 1980, p 808.

Other bioassays which may be used for LHRH antagonists and for thecompounds of the present invention are:

(a) inhibition of LHRH induced FSH and LH release in the rat, in vivo;Vilchez-Martinez, J. A., et al, Endocrinology, 96: 1130 (1975); and,

(b) inhibition of LH and FSH release by dispersed anterior pituitarycell cultures as measured by radioimmunoassay. (Vale, W., et al,Endocrinology 91: 562 (1972).

(c) inhibition of gonadotropin levels in castrated rat and dog (Petrieet al., Male Contraception, Harper and Row, Philadelphia (1985), p.361).

ANTAGONIST EFFECTS AND UTILITIES

The following utilities flow from the antagonist effect of the compoundsherein:

female contraception;

ovulation prevention or delay;

pregnancy termination in domestic animals and pets;

induction of parturition;

synchronization of ovulation;

estrus suppression;

growth promotion in female animals;

luteolysis, menses induction;

therapy for premenstrual syndrome;

therapy for precocious puberty;

therapy for uterine leiomyoma;

early, first trimester abortifacient;

therapy for endometriosis;

therapy for mammary tumors and cysts

therapy for polycystic ovary syndrome/disease;

therapy for uterine carcinoma;

therapy for benign prostatic hypertrophy and for prostatic carcinoma;

male contraception;

therapy for diseases which result from excessive gonadal hormoneproduction in either sex;

functional castration in male food producing animals;

suppression of proestrous bloody discharge in dogs;

diagnostic utilities, such as predisposition to osteoporosis;

prevention of ovarian hyperstimulation;

and other uses as set forth in Vickery, B. H., Endocrine Reviews, 7:115(1986), which is fully incorporated by reference herein.

A particularly interesting use of the instant LHRH antagonists is forthe prevention of ovarian hyperstimulation. Commonly, when a femalesubject suffers from conditions that result in a breakdown of the normalmenstrual cycle, e.g., polycystic ovarian disease, menopausal syndromeresulting from chemotherapy, or oligomenorrhea, fertility can be inducedeither in situ or for in vitro fertilization/egg transfer, byadministration of exogenous gonadotropins. However, this gonadotropictherapy often results in ovarian hyperstimulation and/or multiple birthsdue to the combined effect of the endogenous and exogenousgonadotropins. Accordingly, the LHRH antagonists of this invention areuseful to suppress endogenous gonadropins such that a normal degree ofovarian stimulation can be obtained.

The aspect of the present invention which relates to particular uses forthe above-described compounds is concerned with these utilities, mostparticularly: inhibition of ovulation, treatment of premenstrualsyndrome, treatment of ovarian hyperstimulation due to exogenousgonadotropins, and treatment of endometriosis in the female; inhibitionof spermatogenesis and treatment of prostatic tumors in the male;suppression of isosexual, true (idiopathic) precocious puberty (i.e.,precocious puberty of hypothalamic origin in either male or female);estrus suppression (i.e., interrupting heat in animals); and terminationof pregnancy in animals.

In the practice of the method of this invention an effective amount of acompound of the invention or a pharmaceutical composition containingsame is administered to the subject in need of, or desiring, suchtreatment. These compounds or compositions may be administered by any ofa variety of routes depending upon the specific end use, includingorally, parenterally (including subcutaneous, intramuscular andintravenous administration), vaginally (particularly for contraception),rectally, buccally (including sublingually), transdermally orintranasally. The most suitable route in any given case will depend uponthe use, particular active ingredient, the subject involved, and thejudgment of the medical practitioner. The compound or composition mayalso be administered by means of controlled-release, depot implant orinjectable formulations as described more fully herein below.

In general for the uses herein above described, it is expedient toadminister the active ingredient in amounts between about 0.001 and 5mg/kg body weight. Preferably, for human therapy, the active ingredientwill be administered in the range of from about 0.01 to about 1mg/kg/day; and for animal therapy, the active ingredient will beadministered in the range of from about 0.1 to 1 mg/kg/day. Thisadministration may be accomplished by a single administration, bydistribution over several applications or by slow release in order toachieve the most effective results. Most preferably, for theinterruption of heat or prevention of pregnancy in animals, the dosewill be in the range of from about 1 to 10 mg/kg, administered as asingle dose.

The exact dose and regimen for administration of these compounds andcompositions will necessarily be dependent upon the needs of theindividual subject being treated, the type of treatment, the degree ofaffliction or need and, of course, the judgment of the medicalpractitioner. In general, parenteral administration requires lowerdosage than other methods of administration which are more dependentupon absorption.

A further aspect of the present invention relates to pharmaceuticalcompositions containing as active ingredient a compound of the presentinvention which compositions comprise such compound in admixture with apharmaceutically acceptable, non-toxic carrier. As mentioned above, suchcompositions may be prepared for use for parenteral (subcutaneous,intramuscular or intravenous) administration particularly in the form ofliquid solutions or suspensions; for use in vaginal or rectaladministration particularly in semisolid forms such as creams andsuppositories; for oral or buccal administration particularly in theform of tablets or capsules; or intranasally particularly in the form ofpowders, nasal drops or aerosols.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, PA., 1970.Formulations for parenteral administration may contain as commonexcipients sterile water or saline, alkylene glycols such as propyleneglycol, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. Formulationsfor vaginal or rectal administration, e.g. suppositories, may contain asexcipients, for example, polyalkyleneglycols, vaseline, cocoa butter,and the like. Formulations for nasal administration may be solid andcontain as excipients, for example, lactose or dextran, or may beaqueous or oily solutions for administration in the form of nasal dropsor metered spray. For buccal administration typical excipients includesugars, calcium stearate, magnesium stearate, pregelatinated starch, andthe like.

Nasal administration of the instant nona- and decapeptides isparticularly preferred. The absorption across the nasal mucous membraneis enhanced by surfactant acids, such as for example, glycocholic acid,cholic acid, taurocholic acid, cholanic acid, ethocholic acid,desoxycholic acid, chenodesoxycholic acid, dehydrocholic acid, andglycodeoxy-cholic acid.

One or more surfactant acids or salts, but preferably a singlepharmaceutically acceptable acid salt, can be added to the LHRHantagonist in solution or powder formulation. Suitable pharmaceuticallyacceptable surfactant salts will be those salts which retain thephenomenon of enhanced peptide absorption, as well as the compound'ssurfactant characteristics, and which are not deleterious to the subjector otherwise contraindicated. Such salts are for example those saltsderived from inorganic bases which include sodium, potassium, lithium,ammonium, calcium, magnesium, ferrous, zinc, copper, manganous,aluminum, ferric, manganic salts and the like. Particularly preferredare the ammonium, potassium, sodium, calcium and magnesium salts. Saltsderived from pharmaceutically acceptable organic non-toxic bases includesalts of primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethanolamine,2-dimethylaminoethanol, 2-diethylaminoethanol, tromethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,tromethamine, dicyclohexylamine, choline and caffeine.

More preferably, the surfactant used in the practice of this inventionwill be an alkali metal salt of glycocholic acid, most preferably sodiumglycocholate.

The amount of surfactant used for the practice of this invention will besome amount which increases the absorption of LHRH peptides over that ofother surfactants which also may enhance peptide absorption to a certaindegree. It has been found that such an amount is often in the rangebetween 0.2 and 15%, more often 0.2 to 5 percent by weight/volume of thesolution. It is preferred that the surfactant be present in an amountbetween about 0.5 to 4 percent by weight volume, conveniently about 1percent by weight volume, preferably about 2 percent by weight volume.

Other materials such as preservatives, salts to achieve the tonic valueof tissue, or other additives indicated by known nasal formulationchemistry may be added to these formulations. Particularly advantageousother such materials include surfactants, suitably non-ionic surfactantssuch as the polysorbates, in concentrations suitably in the range 0.1 to5, more suitably 0.25 to 2% weight volume.

It has been found that often to obtain enhanced solubility andstability, the molar ratio of bile acid to peptide is usefully ≧20:1,such as ≧25:1.

It is particularly desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot implant or injectable dosage forms may beutilized. For example, a dosage form may contain a pharmaceuticallyacceptable non-toxic salt of the compound which has a low degree ofsolubility in body fluids, for example, (a) an acid addition salt with apolybasic acid such as phosphoric acid, sulfuric acid, citric acid,tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamicacid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, andthe like; (b) a salt with a polyvalent metal cation such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium and the like, or with an organic cation formed from e.g.,N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, may be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection or implantation would contain the compound or salt dispersedor encapsulated in a slowly degrading, non-toxic, non-antigenic polymersuch as a polylactic acid/polyglycolic acid polymer. The compounds or,preferably, relatively insoluble salts such as those described above mayalso be formulated in cholesterol matrix pellets, or silastomer matriximplants, particularly for use in animals. Additional slow release,depot implant or injectable formulations, e.g. liposomes, are well knownin the literature. See, for example, Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., NewYork, 1978. Particular reference with respect to LHRH type compounds maybe found, for example, in U.S. Pat. No. 4,010,125.

SYNTHESIS OF THE PEPTIDES

The polypeptides of the present invention may be synthesized by anytechniques that are known to those skilled in the peptide art. Anexcellent summary of the many techniques so available may be found in J.M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. FreemanCo., San Francisco, 1969, and J. Meienhofer, Hormonal Proteins andPeptides, Vol. 2, p. 46., Academic Press (New York), 1973 for solidphase peptide synthesis and E. Schroder and K. Lubke, The Peptides, Vol.1, Academic Press (New York), 1965 for classical solution synthesis.

In general, these methods comprise the sequential addition of one ormore amino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid can then be either attached to an inert solidsupport or utilized in solution by adding the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected, under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is then added, andso forth. After all the desired amino acids have been linked in theproper sequence, any remaining protecting groups (and any solid support)are removed sequentially or concurrently, to afford the finalpolypeptide. By simple modification of this general procedure, it ispossible to add more than one amino acid at a time to a growing chain,for example, by coupling (under conditions which do not racemize chiralcenters) a protected tripeptide with a properly protected dipeptide toform, after deprotection, a pentapeptide.

PREFERRED EMBODIMENTS OF SYNTHESIS

A particularly preferred method of preparing compounds of the presentinvention involves solid phase peptide synthesis.

In this particularly preferred method the α-amino function of the aminoacids is protected by an acid or base sensitive group. Such protectinggroups should have the properties of being stable to the conditions ofpeptide linkage formation, while being readily removable withoutdestruction of the growing peptide chain or racemization of any of thechiral centers contained therein. Suitable protecting groups aret-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like,especially t-butyloxycarbonyl (Boc).

Particularly preferred side chain protecting groups are, for arginine:nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc andadamantyloxycarbonyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl,2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; forserine: benzyl and tetrahydropyranyl; for histidine: benzyl,p-toluenesulfonyl and 2,4-dinitrophenyl.

The C-terminal amino acid is attached to a suitable solid support.Suitable solid supports useful for the above synthesis are thosematerials which are inert to the reagents and reaction conditions of thestepwise condensation-deprotection reactions, as well as being insolublein the media used. Suitable solid supports arechloromethylpolystyrene-divinylbenzene polymer,hydroxymethyl-polystyrene-divinylbenzene polymer, and the like,especially chloromethyl-polystyrene-1% divinylbenzene polymer. For thespecial case where the C-terminus of the compound will be glycinamide, aparticularly useful support is thebenzhydrylamino-polystyrene-divinylbenzene polymer described by P.Rivaille, et al, Helv. Chim. Acta., 54, 2772 (1971). The attachment tothe chloromethyl polystyrene-divinylbenzene type of resin is made bymeans of the reaction of the N.sup.α -protected amino acid, especiallythe Boc-amino acid, as its cesium, tetramethylammonium,triethylammonium, 1,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt inethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like,especially the cesium salt in DMF, with the chloromethyl resin at anelevated temperature, for example between about 40° and 60° C.,preferably about 50° C., for from about 12 to 48 hours, preferably about24 hours. The N.sup.α -Boc-amino acid is attached to the benzhydrylamineresin by means of an N,N'-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HBT) mediated coupling for from about 2 toabout 24 hours, preferably about 12 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or DMF, preferably dichloromethane. The coupling ofsuccessive protected amino acids can be carried out in an automaticpolypeptide synthesizer as is well known in the art. The removal of theN.sup.α -protecting groups may be performed in the presence of, forexample, a solution of trifluoroacetic acid in methylene chloride,hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or otherstrong acid solution, preferably 50% trifluoroacetic acid indichloromethane at about ambient temperature. Each protected amino acidis preferably introduced in approximately 2.5 molar excess and thecoupling may be carried out in dichloromethane, dichloromethane/DMFmixtures, DMF and the like, especially in methylene chloride at aboutambient temperature. The coupling agent is normally DCC indichloromethane but may be N,N'-di-iso-propylcarbodiimide (DIC) or othercarbodiimide either alone or in the presence of HBT,N-hydroxysuccinimide, other N-hydroxyimides or oximes. Alternately,protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected polypeptideis removed from the resin. When the linkage to the resin support is ofthe benzyl ester type, cleavage is by means of aminolysis with analkylamino or fluoroalkylamine for peptides with a proline C-terminus,or by aminolysis with, for example, ammonia/methanol or ammonia/ethanolfor peptides with a glycine C-terminus at a temperature between about10° and 50° C., preferably about 25° C., for between about 12 and 24hours preferably about 18 hours. Alternatively, the peptide may beremoved from the resin by transesterification, e.g., with methanol,followed by aminolysis. The protected peptide may be purified at thispoint by silica gel chromatography. The removal of the side chainprotecting groups from the polypeptide is performed by treating theaminolysis product with, for example, anhydrous liquid hydrogen fluoridein the presence of anisole or other carbonium scavenger, treatment withhydrogen fluoride/pyridine complex, treatment withtris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction withhydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride, and anisole at a temperature between about -10° and +10° C.,preferably about 0° C., for between about 15 minutes and 1 hour,preferably about 30 minutes. For the glycine terminal peptides on thebenzhydrylamine resins, the resin cleavage and deprotection steps may becombined in a single step utilizing liquid hydrogen fluoride and anisoleas described above. The fully deprotected polypeptide is then purifiedby a sequence of chromatographic steps employing any or all of thefollowing types: ion exchange on a weakly basic resin in the acetateform; hydrophobic adsorption chromatography on underivatizedpolystyrene-divinylbenzene (for example Amberlite XAD); silica geladsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g., on SephadexG-25, or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

If a racemic amino acid is used in the 1, 2, 3 or 6 position, thediastereomeric nonapeptide or decapeptide final products are separated,and the desired peptide containing a D-amino acid in the appropriateposition is isolated and purified, preferably during the above-describedchromatographic process.

The preparation of peptides having C-terminal azaglycine amides ispreferably done using classical peptide solution synthesis using knownpeptide intermediates.

Thus, in another aspect the present invention relates to a method forpreparing compounds of the invention and of the pharmaceuticallyacceptable salts thereof which process comprises:

removing protecting groups and, optionally, covalently bound solidsupport from a protected polypeptide to afford a compound of Formula (I)or a salt thereof, and optionally

(a) converting a compound of Formula (I) to a pharmaceuticallyacceptable salt, or

(b) converting a salt of a compound of Formula (I) to a pharmaceuticallyacceptable salt, or

(c) decomposing a salt of a compound of Formula (I) to a freepolypeptide of Formula (I).

The following examples are given to enable those skilled in the art tomore fully understand and practice the present invention. They shouldnot be construed as a limitation upon the scope of the invention, butmerely as being illustrative and representative thereof.

PREPARATION A 3-(2-Naphthyl)-D,L-Alanine

The preparation of 3-(2-naphthyl)-D,L-alanine is carried out accordingto the procedure set out in U.S. Pat. No. 4,341,767.

Preparation of N-acetyl-3-(2-naphthyl)-D,L-alinine, its conversion tomethyl N-acetyl-3-(2-naphthyl)-D,L-alaninate, and separation of the Disomer is carried out by the procedure disclosed in U.S. Pat. No.4,341,767.

PREPARATION B Benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-diisopropyl-D-homoargininatetoluenesulfonate

A mixture of 5.24 g of benzyl N.sup.α -benzyloxycarbonyl-D-lysinatetoluenesulfonate (B. Bezus and L. Zervas, J. Am. Chem. Soc. 83, 719(1961)) and 1.72 ml of diisopropylethylamine in 60 ml of dioxane istreated with 1.89 g of N,N'-diisopropylcarbodiimide. The reactionmixture is stirred at 100° C. for 6 hours, cooled to room temperatureand concentrated to a solid. The solid is suspended in 20 ml of warmDMF, filtered to remove N,N'-diisopropylurea and the filtrateconcentrated to a solid. Benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-diisopropyl-D-homoargininatetoluenesulfonate is obtained as a white solid by crystallization frommethanol/ethyl acetate [α]_(D) -7.26° (C 0.3, MeOH).

Similarly, by using the above procedure, but substituting:

N,N'-dicyclohexylcarbodiimide;

N,N'-di-n-hexylcarbodiimide;

N,N'-diethylcarbodiimide;

N,N'-di-n-propylcarbodiimide;

N-i-propylcarbodiimide;

N-propylcarbodiimide;

N-n-butylcarbodiimide;

N,N'-di-n-butylcarbodiimide;

N,N'-dimethylcarbodiimide;

N,N'-di-i-butylcarbodiimide;

N,N'-di-n-pentylcarbodiimide;

N,N'-di-i-pentylcarbodiimide;

N,N'-diphenylcarbodiimide;

N,N'-ditolylcarbodiimide; or

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-HCl; and the like, thereare obtained:

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-dicyclohexyl-D-homoargininate, [α]_(D)8.07° (C 0.9 MeOH);

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-diethyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-n-propyl-D-homoargininate [α]_(D)8.07° (C 0.9 MeOH);

benzyl N.sup.α -benzyloxycarbonyl-N-guanidino-n-propyl-D-homoargininate;

benzyl N.sup.α -benzyloxycarbonyl-N-guanidino-n-butyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-n-butyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-i-butyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-n-pentyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-phenyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-dimethyl-D-homoargininate;

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-n-hexyl-D-homoargininate; and

benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-di-isopropyl-D-argininate, [α]_(D)-10.5° (C 0.5, MeOH); as their benzenesulfonate salts. Similarly, bysubstituting benzyl N.sup.α -benzyloxycarbonyl-D-ornithinate for theD-lysinate there may be obtained the corresponding arginine analogs astheir toluenesulfonate salts.

PREPARATION C Benzyl N.sup.α -benzyloxycarbonyl-N^(G),N^(G')-ethano-D-homoargininate

To a mixture of 15 ml of toluene and 15 ml of t-BuOH was added 2.71 g ofbenzyl N.sup.α -benzyloxycarbonyl-lysinate and 1.46 g of2-methylthioimidazoline.HI (available from Aldrich). The pH of themixture was brought to about 8 by the addition of diisopropylethylamineand the solution was heated under reflux for 24 hours.

The solution was concentrated in vacuo and the residue was loaded on asilica gel column (250 g). The column was eluted with a gradient fromCH₂ Cl₂ /MeOH (19:1) to CH₂ Cl₂ /MeOH (7:3). The fractions containingproduct were determined by TLC, pooled, and concentrated to dryness, 2.9g of white foam.

A 2 g portion of the above-named product was dissolved in 50 ml of EtOHcontaining 0.8 g of 10% Pd/C. The solution was stirred under H₂ for 8hours. The mixture was filtered on celite and the filtrate wasconcentrated to dryness to give N^(G),N^(G') -ethano-homoarginine as awhite foam, 1.2 g.

In a similar manner, but employing S-methyl3,4,5,6-tetrahydro-2-pyrimidinethiol (Aldrich) as the guanylatingspecies, was obtained N^(G),N^(G') -propano-homoarginine as a whitefoam.

In a similar manner, but employing S-methylbis-(2,2,2-trifluoroethyl)-thiouronium iodide as the guanylatingspecies, there was obtained N^(G),N^(G')-bis(trifluoroethyl)-homoarginine (Bth) as a white foam.

PREPARATION D N.sup.α-t-butyloxycarbonyl-N,N'-guanidino-diisopropyl-D-homoargininetoluenesulfonate

This Preparation illustrates the preparation of N.sup.α-t-butyloxycarbonyl derivatives ofN,N'-guanidino-disubstituted-D-homoarginines from their toluenesulfonateprecursors.

A mixture of benzyl N.sup.α-benzyloxycarbonyl-N,N'-guanidino-diisopropyl-D-homoargininatetoluenesulfonate (3.25 g) and 100 mg of 10% Pd/C in 50 ml of glacialacetic acid is treated with hydrogen gas at atmospheric pressure for 4hours. The catalyst is filtered on celite and the filtrate isconcentrated to a solid, N,N'-guanidino-diisopropyl-D-homoargininetoluenesulfonate. A solution of this compound (2.13 g) in 60 ml of 50%dioxane/water is treated with 10 ml of 1N sodium hydroxide and 0.4 g ofmagnesium oxide. This mixture is then treated with 1.1 g ofdi-t-butyldicarbonate and stirred at room temperature for 2 hours. Themagnesium salt is filtered and the filtrate is concentrated undervacuum. The basic solution is washed with ethanol, then brought to pH2.5 with sodium sulfate. The acidic aqueous solution is extracted withethylacetate which is dried over magnesium sulfate. The drying agent isfiltered and the filtrate is concentrated. Crystallization from ethylacetate/hexane affords N.sup.α-t-butyloxycarbonyl,N,N'-guanidino-diisopropyl-D-homoargininetoluenesulfonate.

Proceeding in a similar manner, but substituting the appropriatetoluenesulfonate precursors, other N.sup.α-t-butyloxycarbonyl-N,N'-guanidino-disubstituted-D-homoarginine orD-arginine compounds may be prepared.

PREPARATION E N.sup.α-t-butyloxycarbonyl-3-(4'-(1'-propylpiperidyl))-D-alanine

A 4.6 g portion of sodium metal was added to 400 ml of absolute ethanoland heated. To the resultant solution of sodium ethoxide was added 21.7g of diethyl acetamidomalonate and 16.4 g of 4-picolyl chloridehydrochloride (Aldrich Chem. Co.). The reaction mixture was heated to100° C. for 4 hours, cooled, filtered and concentrated in vacuo. Themixture was loaded on a silica gel column in methylene chloride/methanol(19:1) and eluted with the same mixture. The product was located as afast running UV positive spot by TLC on silica gel in methylenechloride/methanol (19:1). Combined fractions were concentrated toprovide the product.

The product from the foregoing paragraph was dissolved in 200 ml ofethanol and treated with a solution of 2.72 g of sodium hydroxide in 40ml of water at 50° C. for 6 hours. The solution was acidified with 12 mlof 6N HCl, concentrated to dryness and taken up in 200 ml of dioxane.The suspension was filtered and the filtrate heated at reflux for 2hours. The solution was cooled and concentrated to dryness to yieldethyl N.sup.α -acetyl-3-(4-pyridyl)-D,L-alanine as a white solid.

A portion of this N-acetyl ester was resolved by treatment with 200 mlof the enzyme subtilisin Carlsberg (Sigma Chem. Co., protease VIII) in amixture of 300 ml of dimethyl sulfoxide and 400 ml of 0.01M KCl (pH7.2). The pH was maintained by addition of 1N NaOH on a pH stat. After a6 hour period, the resolution was complete. The solution was dilutedwith 400 ml of water and extracted with 4×750 ml of ethyl acetate. Theorganic layers were combined and dried over magnesium sulfate andconcentrated to yield ethyl N.sup.α -acetyl-3-(4-pyridyl)-D-alaninate asan oil.

The oil was reacted with 1.22 g of n-propyl bromide in 50 ml of ethanolafter which the solution was concentrated to dryness to yield ethylN.sup.α -acetyl-3-(1-propyl-pyridinium-4-yl)-D-alininate bromide as awhite hygroscopic solid.

This white solid was dissolved in 200 ml of ethanol and was reducedunder an atmosphere of hydrogen gas using 100 mg of 10% Pd/C as acatalyst. After an 18 hour reduction period, the catalyst was filteredout and the solutin concentrated to yield ethyl N.sup.α-acetyl-3-(4'-(1'-propylpiperidyl))-D-alininate as a tan solid. The freeacid was prepared by refluxing the ethyl ester in 100 ml of 6N HCl for 4hours to yield 3-(4'-(1'-propylpiperidyl))-D-alanine as a white solid.

The free acid was dissolved in 100 ml of dioxane/water (1:1) and treatedwith 2 g of di-t-butyldicarbonate. The pH was maintained at 9 byaddition of 1N NaOH on a pH stat. After 2 hours the reaction mixture wasconcentrated in vacuo, washed with 100 ml of ethyl ether and the aqueouslayer was loaded on an Amberlite XAD-2 hydrophobic resin. The column waseluted with 250 ml of water followed by 250 ml of 50% ethanol/water. Theethanol eluate was pooled and concentrated to dryness to yield N.sup.α-t-butyloxycarbonyl-3-(4'-(1'-propylpiperidyl))-D-alanine as a whitesolid.

Proceeding in similar manner, but substituting 3-picolyl chloridehydrochloride for 4-picolyl chloride hydrochloride, there is preparedN.sup.α -t-butyloxycarbonyl-3-(3'-(1'-propylpiperidyl))-D-alanine.

PREPARATION F Boc-Gly-O-Resin

4.9 g of Boc-glycine was dissolved in a mixture of 50 ml. ethanol and 50ml. distilled water. The pH of the solution was brought to 7 withaqueous cesium bicarbonate. The solvent was then removed under vacuum.

After 18 hours of drying under high vacuum, the residue was dissolved in150 ml. dry DMF. 25 g chloromethylated polystyrene--1% divinylbenzene(Merrifield) resin (corresponding to 25 mmole chloride) was added. Themixture was shaken at 50° C. for 24 hours, filtered, and the resin wasthen washed sequentially with DMF, water, and ethanol. The resin wasdried under vacuum for 3 days to yield 28.34 g of Boc-Gly-O-Resin.

PREPARATION G A. S-Methyl 3,4,5,6-tetrahydro-2-pyrimidinethio.hydroiodide

A solution of 23.24 g of 3,4,5,6-tetrahydro-2-pyrimidine thiol in 175 mlof MeOH was treated with 15.57 ml of MeI and refluxed for 1.5 hr. Thesolvent was evaporated under vacuum and the residue suspended in Et₂ O.The precipitate was filtered and dried in vacuo to provide S-Methyl3,4,5,6-tetrahydro-2-pyrimidine thiol as 51.4 g of white crystals.

B. N.sup.α -t-Butyloxycarbonyl-N^(G),N^(G') -propano-L-homoarginine

S-Methyl 3,4,5,6-tetrahydro-2-pyrimidine thiol was sprung from 51.4 g ofits HI salt by partitioning between 300 ml of CH₂ Cl₂ and 50 ml of 4NNaOH. The resulting CH₂ Cl₂ solution was evaporated to ˜100 ml and ˜100ml of EtOH was added. A solution of 24 g of lysine hydrochloride in 66ml of 2N NaOH was treated at 60° C. in a dropwise fashion with thesolution of S-methyl 3,4,5,6-tetrahydro-2-pyrimidine thiol. Stirring wascontinued overnight at 60° C. under N₂. An additional 10.78 g of the HIsalt was sprung with 15 ml of 4N NaOH, extracted with 90% of CH₂ Cl₂ andadded dropwise with stirring for another 24 hour period at 60° C. atwhich time the reaction was essentially complete.

The reaction mixture was washed with 2 portions of EtOAc and the aqueouslayer containing Pha was diluted with 200 ml of dioxane and 200 ml of H₂O. The solution was cooled to 0° C. before addition of 33 g ofdi-t-butyldicarbonate and 6 g of MgO. An additional batch of 4 g of MgOand 22 g of di-t-butyldicarbonate pushed the reaction to completion.

The MgO was filtered on celite and the filtrate was evaporated in vacuoto 1/2 volume. The residue was washed twice with Et₂ O before loading ona silica gel column (750 g silica gel packed in CH₃ CN). The column waswashed with 5 L CH₃ CN, eluted with 10% H₂ O/CH₃ CN (2 L), and furthereluted with 20% H₂ O/CH₃ CN (5 L). The product fractions were located bythin layer chromatography (CN₃ CN/HOAc/H₂ O; 4:1:1) on silica gelplates. The product fractions were pooled and concentrated to yield theproduct as 5.04 g of white foam of m.p. 96° C., [α]_(D) ²⁵ 16.1° C. (C0.54, MeOH), and an additional 15 g of slightly impure oil.

In a similar fashion but substituting the appropriate S-MethylN,N'-dialkylthiouronium hydroiodides are obtained the correspondingN.sup.α -t-butyloxycarbonyl-N^(G),N^(G') -dialkylhomoarginines.

EXAMPLE 1 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH₂ CF₃)₂-D-Tyr-Leu-hArg(CH₂ CF₃)₂ -Pro-D-Ala-NH₂

The title compound is also represented herein asN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-Ala-NH₂.

In the reaction vessel of a Beckman 990 Peptide Synthesizer was placed0.758 g. (0.5 mmol.) of benzhydrylamino-polystyrene resin (PeninsulaLabs, 0.66 mmol/g). The first amino acid was coupled by addition of0.284 g of Boc-D-Ala-OH, 0.202 g of HBt and 3 ml of 0.5Mdiisopropylcarbodiimide. After a 3 hr coupling period and washing of theresin, further amino acids were added sequentially to this resin bymeans of a synthesis program, as follows:

    ______________________________________                                         Step                                                                         ______________________________________                                        1    CH.sub.2 Cl.sub.2 wash                                                                            1 time   1.5 min                                     2    50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2 --                                                      1 time   1.5 min                                          deprotection                                                             3    50% CF.sub.3 CO.sub.2 H/CH.sub.2 Cl.sub.2 --                                                      1 time   30 min                                           deprotection                                                             4    CH.sub.2 Cl.sub.2 wash                                                                            3 times  1.5 min                                     5    10% triethylamine/CH.sub.2 Cl.sub.2                                                               2 times  1.5 min                                     6    CH.sub.2 Cl.sub.2 wash                                                                            3 times  1.5 min                                     7    N.sup.α --Boc-amino acid                                                                    1 time   add                                              solution in 50% CH.sub.2 Cl.sub.2 /DMF                                   8    N,N'--diisopropylcarbo-                                                                           1 time   add                                              diimide solution (0.5 M)                                                 9    CH.sub.2 Cl.sub.2 rinse and hold-                                                                 1 time   coupling                                         coupling                     reaction                                                                      2 hr                                        10   CH.sub.2 Cl.sub.2 --rinse add                                                                     1 time   1.5 min                                     11   CH.sub.2 Cl.sub.2 wash                                                                            3 times  1.5 min                                     12   ethanol wash        3 times  1.5 min                                     13   CH.sub.2 Cl.sub.2 wash                                                                            3 times  1.5 min                                     ______________________________________                                    

Steps 1-13 complete a coupling cycle for one amino acid and completenessof the reaction may be checked by the ninhydrin method of E. Kaiser, etal., Anal. Biochem., 34, 595 (1970).

The resin was coupled sequentially with a 2.0 to 3.0 molar excess ofeach protected amino acid and DIC. Thus, the resin was treated duringsuccessive coupling cycles with

0.269 g. Boc-Pro-OH,

0.610 g. Boc-Bth-OH,

0.311 g. Boc-Leu-OH.H₂ O

0.375 g. Boc-D-Tyr-OH,

0.610 g. Boc-Bth-OH,

0.380 g. Boc-Ser(Benzyl)-OH,

0.320 g. Boc-D-Pal(3)-OH,

0.390 g. Boc-D-pCl-Phe-OH,

0.400 g. Boc-D-Nal(2)-OH, and

2.5 ml. acetic anhydride.

The resin was removed from the reaction vessel, washed with CH₂ Cl₂, anddried in vacuo to yield 1.43 g. of protected polypeptide resin. Theprotected peptide was deprotected and removed from the resin bytreatment with 25 ml. anhydrous liquid HF in the presence of 2.5 ml. ofanisole (scavenger) in a Kel-F reaction vessel at 0° C. for 1 hr. The HFwas evaporated under vacuum and the residue ofN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH₂ CF₃)₂ -D-Tyr-Leu-hArg(CH₂CF₃)₂ -Pro-D-Ala-NH₂, as its HF salt, was washed with ether. The residuewas then extracted with glacial acetic acid (3×30 ml). The acetic acidextract was evaporated to dryness. The crude material was converted tothe acetate salt by passage in water through a column of AG3 (a weaklybasic tertiary amine resin) which had been converted to the acetateform. Lyophilization of the eluate yielded 0.5 g. of the crude peptideacetate salt as a white solid.

The crude peptide was purified by high performance liquid chromatographyon a 2.5×100 cm. column of Licroprep RP-18 (25-40 micron) equilibratedto the running buffer 50% CH₃ CN/50% H₂ O (0.1% in CF₃ CO₂ H, pH 2.5).The major UV absorbing (280 nm) peak eluting at approximately 2 columnvolumes was collected, concentrated to dryness, and lyophilized 3 timesfrom distilled water to yield 64 mg of pureN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH₂ CF₃)₂ -D-Tyr-Leu-hArg(CH₂CF₃)₂ -Pro-D-Ala-NH₂, [α]_(D) ²⁵ =-17.96° (C 0.4, HOAc).

B. Proceeding in a similar manner but substituting the appropriate A, B,C, D, E, F, G or J amino acid for those recited, there were prepared thecorresponding decapeptides exemplified below:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;and

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂.

C. Proceeding in a similar manner but substituting the appropriate A, B,C, D, E, F, G or J amino acid for those recited, there are prepared thecorresponding decapeptides exemplified below:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Deh-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pha-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Nal(2)-Leu-Deh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal(2)-Leu-Mbh-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Nal(2)-Leu-Bth-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Nal(2)-Leu-Pha-Pro-D-AlaNH₂ ;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Deh-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2;

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Pha-Pro-D-AlaNH.sub.2; and

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH.sub.2.

EXAMPLE 2 N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH₂ CF₃)₂-D-Tyr-Leu-hArg(CH₂ CF₃)₂ -Pro-NHEt

For the synthesis of analogs with a C-terminal Pro-NHCH₂ CH₃, asynthesis program identical to that described in Example 1 is used. TheBeckman 990 Synthesizer reaction vessel is loaded with 0.71 g. ofBoc-Pro-O-Resin, prepared by the reaction of 1.3 molar excess of the drycesium salt of Boc-Pro-OH with chloromethyl-polystyrene/1%divinylbenzene (Lab Systems, Inc.). The quantity of Boc-Pro-O-Resintaken contains 0.5 mmol. of proline.

The resin is coupled sequentially with a 2.0 to 3.0 molar excess of eachprotected amino acid and DIC. Thus, the resin is reacted duringsuccessive coupling cycles with

0.610 g. Boc-Bth-OH,

0.311 g. Boc-Leu-OH.H₂ O

0.375 g. Boc-D-Tyr-OH,

0.610 g. Boc-Bth-OH,

0.380 g. Boc-Ser(Benzyl)-OH,

0.320 g. Boc-D-Pal(3)-OH,

0.390 g. Boc-D-pCl-Phe-OH,

0.400 g. Boc-D-Nal(2)-OH, and

2.5 ml. acetic anhydride.

The resin is removed from the reaction vessel, washed with CH₂ Cl₂, anddried in vacuo to yield 1.5 g. of protected polypeptide resin.

The protected polypeptide is cleaved from the resin by aminolysis with25 ml. of ethylamine for 18 hours at 2° C. The ethylamine is allowed toevaporate and the resin is extracted with methanol. The methanol isevaporated to yield 0.7 g. of protected peptide. This protected peptideis mixed with 2.5 ml of anisole and 25 ml of redistilled (from CoF₃)anhydrous liquid HF at 0° C. for 1 hour in a Kel-F reaction vessel. TheHF is evaporated under vacuum and the residue is washed with ether. Theresidue is dissolved in 2M acetic acid and converted to the acetate saltby passage in water through a column of AG3 which had been converted tothe acetate form. Lyophilization of the eluate yields 0.5 g. of thecrude peptide acetate salt as a white solid. Final purification isachieved by preparative high performance liquid chromatography on a2.5×100 mm. column of 40-50 micron octadecylsilylated silica (Merck,Lichroprep C₁₈) using 50% CH₃ CH (0.1% CF₃ CO₂ H) as eluate.Lypophilization of the product from H₂ O yieldsN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-hArg(CH₂ CF₃)₂ -D-Tyr-Leu-hArg(CH₂CF₃)₂ -ProNHEt as 70 mg of white powder.

Proceeding in a similar manner, but substituting the required protectedamino acid residues where appropriate, there are prepared the followingcompounds:

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-NHEt,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-NHEt,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-NHEt,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-NHEt,

N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Nal(2)-Leu-Bth-Pro-NHEt.

Repeating the above cleavage, substituting a stoichiometric amount ofmethylamine and propylamine for ethylamine there are obtained thecorresponding methylamides or propylamides of the aforementionednonapeptides.

EXAMPLE 3 Preparation of Salts

A. A solution of 0.1 g of the hydrogen fluoride salt of (N-Ac-D-Nal(2)¹,D-pCl-Phe², D-Pal(3)³,6, Bth⁸, D-Ala¹⁰)LHRH (See Example 1) is dissolvedin 50 ml of water and passed through a column of 50 g Dowex 3 anionexchange resin which had previously been equilibrated with acetic acidand washed with deionized water. The column is eluted with deionizedwater and the effluent is lyophilized to yield the corresponding aceticacid salt of (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Pal(3)³,6, Bth⁸,D-Ala¹⁰)LHRH.

Repeating the above, substituting other acids for acetic acid during theequilibration of the resin, there may be obtained, for example, thecorresponding salts with hydrochloric acid, hydrobromic acid, sulfuricacid, phosphoric acid, nitric acid, benzoic acid, and the like.

Similarly there may be prepared the acid addition salts of the otherpeptides analogous to LHRH, described herein.

B. In the case of salts of low water solubility, these may be preparedby precipitation from water utilizing the desired acid. For example:

Zinc tannate salt--a solution of 10 mg of (N-Ac-D-Nal(2)¹, D-pCl-Phe²,D-Pal(3)³,6, Bth⁸, D-Ala¹⁰)LHRH acetic acid salt in 0.1 ml of water wastreated with a solution of 8 mg of tannic acid in 0.08 ml of 0.25M NaOH.A solution of 5 mg of ZnSO₄ heptahydrate in 0.1 ml of water wasimmediately added to the solution of the LHRH analog.

The resultant suspension was diluted with 1 ml water and the precipitatewas centrifuged. The supernatant was decanted and the residue was washedtwice with 1 ml portions of water by centrifugation of the precipitateand decantation of the supernatant. The precipitate was dried in vacuoto yield 15 mg of the mixed zinc tannate salt of the above named LHRHanalog.

Pamoate salt--to a solution of 50 mg (N-Ac-D-Nal(2)¹, D-pCl-Phe²,D-Pal(3)³,6, Bth⁸, D-Ala¹⁰)LHRH acetic acid salt in a mixture of 1.6 mlof ethanol and 0.1 ml of 0.25M NaOH was added a solution of 11 mg ofpamoic acid in 0.3 ml of 0.25M NaOH. The solvents were removed atreduced pressure and the residue was suspended in 2 ml of water,centrifuged, and the supernatant was decanted. The precipitate waswashed with 1.5 ml H₂ O, centrifuged, and the supernatant was decanted.The precipitate was dried in vacuo to yield 54 mg of the pamoate salt ofthe above named LHRH analog.

In a similar manner other salts of low water solubility may be prepared.

C. Preparation of acid addition salt from free peptide.

To a solution of 50 mg of (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Pal(3)³,6,Bth⁸, D-Ala¹⁰)LHRH as the free base is added 30 ml of 1N acetic acid.The resulting solution is lyophilized to yield 50 mg of the acetic acidsalt of the above.

Similarly, replacing acetic acid with other acids (in stoichiometricallyequivalent amounts relative to peptide) there are obtained other acidaddition salts of the peptides herein, for example, the salts withhydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid.

D. Preparation of salt with metal cation, e.g., zinc salt.

To a solution of 50 mg (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Pal(3)³,6, Bth⁸,D-Ala-¹⁰)LHRH acetic acid salt in a mixture of 0.4 ml of 0.25M NaOH, 0.3ml water, and 1 ml ethanol was added a solution of 15 mg of ZnSO₄heptahydrate in 0.2 ml of water. The precipitate was centrifuged and thesupernatant was decanted. The precipitate was washed with 1 ml of waterby centrifugation and decantation of the supernatant. The precipitatewas dried in vacuo to yield the zinc salt of the above named LHRHanalog.

In a similar manner salts with other multivalent cations e.g. calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmiumand the like, may be prepared.

EXAMPLE 4 Conversion of Salts to Free Base

A solution of 50 mg of (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Pal(3)³,6, Bth⁸,D-Ala¹⁰)LHRH acetic acid salt in 25 ml. of water is passed through a 50g column of Dowex 1 (strongly basic, quaternary ammonium anion exchangeresin) which had been equilibrated with NaOH solution to make thecounter ion hydroxide. The column is eluted with 150 ml of water and theeluant is lyophilized to yield 45 mg of the corresponding polypeptide asthe free base.

Similarly other acid addition salts of compounds of the peptides herein,e.g., those mentioned in Example 6, may be converted to thecorresponding free bases.

EXAMPLE 5 Pharmaceutical Formulations

The following are typical pharmaceutical compositions containing, asactive ingredient, an LHRH antagonist of the present invention, forexample (N-Ac-D-Nal(2)¹, D-pCl-Phe², D-Pal(3)³,6, Bth⁸, D-Ala¹⁰)LHRH, byitself or as a pharmaceutically acceptable salt, e.g., the acetic acidaddition salt, the zinc salt, the zinc tannate salt, etc.

    ______________________________________                                        A. Tablet Formulations                                                        ______________________________________                                        1.       LHRH Antagonist      10.0   mg                                                Compressible Sugar, USP                                                                            86.0   mg                                                Calcium Stearate     4.0    mg                                       2.       LHRH Antagonist      10.0   mg                                                Compressible Sugar, USP                                                                            88.5   mg                                                Magnesium Stearate   1.5    mg                                       3.       LHRH Antagonist      5.0    mg                                                Mannitol, USP        83.5   mg                                                Magnesium Stearate, USP                                                                            1.5    mg                                                Pregelatinized Starch, USP                                                                         10.0   mg                                       4.       LHRH Antagonist      10.0   mg                                                Lactose, USP         74.5   mg                                                Pregelatinized Starch, USP                                                                         15.0   mg                                                Magnesium Stearate, USP                                                                            1.5    mg                                       ______________________________________                                    

METHOD OF MANUFACTURE

(a) LHRH Antagonist is dissolved in water, a sufficient quantity to forma wet granulation when mixed with the sugar portion of the excipients.After complete mixing, the granulation is dried in a tray or fluid-beddryer. The dry granulation is then screened to break up any largeaggregates and then mixed with the remaining components. The granulationis then compressed on a standard tabletting machine to the specifictablet weight.

(b) In this manufacturing method, all formulations would include 0.01%gelatin, USP. The gelatin would be first dissolved in the aqueousgranulation solvent followed by the LHRH analog. The remaining steps areas in (a) above.

Formulation 4 could also be used as a tablet for oral administration.

    ______________________________________                                        B. Long Acting Intramuscular Injectable Formulation                           1. Long Acting I.M. Injectable - Sesame Oil Gel                               ______________________________________                                        LHRH Antagonist         10.0   mg                                             Aluminum monostearate, USP                                                                            20.0   mg                                             Sesame oil q.s. ad      1.0    ml                                             ______________________________________                                    

The aluminum monostearate is combined with the sesame oil and heated to125° C. with stirring until a clear yellow solution forms. This mixtureis then autoclaved for sterility and allowed to cool. The LHRH analog isthen added aseptically with trituration. Particularly preferred LHRHanalogs are salts of low solubility, e.g. zinc salts, zinc tannatesalts, pamoate salts, and the like. These exhibit exceptionally longduration of activity.

    ______________________________________                                        2. Long Acting I.M. Injectable - Biodegradable                                Polymer Microcapsules                                                         ______________________________________                                        LHRH Antagonist     7%                                                        25/75 glycolide/lactide                                                                          93%                                                        copolymer (0.5 intrinsic                                                      viscosity)                                                                    ______________________________________                                    

Microcapsules of above formulation suspended in:

    ______________________________________                                        Dextrose         5.0%                                                         CMC, sodium      0.5%                                                         Benzyl alcohol   0.9%                                                         Tween 80         0.1%                                                         Water, purified q.s.                                                                           100.0%                                                       ______________________________________                                    

25 mg of microcapsules would be suspended in 1.0 ml of vehicle.

    ______________________________________                                        C. Aqueous Solution for Intramuscular Injection                               ______________________________________                                        LHRH Antagonist          0.5%                                                 Acetic Acid              0.02 M                                               Benzyl Alcohol           0.9%                                                 Mannitol                 3.5%                                                 Propylene Glycol         20%                                                  NaOH sufficient to adjust pH to                                                                        5                                                    Water q.s. to            100%                                                 ______________________________________                                    

Acetic acid, benzyl alcohol, mannitol and propylene glycol weredissolved in 90% of the water. Then the Antagonist was dissolved in thissolution, and the pH adjusted with NaOH. Water was added to q.s. Thesolution was filtered through a one micron filter, packaged into vials,and sterilized by autoclaving.

    ______________________________________                                        D. Aqueous Formulation for Nasal Administration                               LHRH Antagonist         50     mg                                             0.02 M Acetate Buffer   5      ml                                             Sodium Glycocholate     500    mg                                             0.02 Acetate Buffer, pH 5.2 q.s.                                                                      10     ml                                             E. Formulation for Rectal Administration                                      Suppository Vehicle:                                                          LHRH Antagonist          5.0   mg                                             Witepsol H15            20.0   gm                                             ______________________________________                                    

The LHRH antagonist is combined with the molten Witepsol H15, mixed welland poured into 2 gm molds.

What is claimed is:
 1. A compound of the formula ##STR6## or apharmaceutically acceptable salt thereof, wherein: A is an amino acylresidue selected from the group consisting of either the D- or theL-isomer of: N-Ac-D,L-Δ³,4 -prolyl, N-Ac-D,L-prolyl,N-Ac-D,L-phenylalanyl, N-Ac-D,L-p-chlorophenylalanyl,N-Ac-D,L,-p-fluorophenylalanyl, N-Ac-3-(1-naphthyl)-D,L-alanyl,N-Ac-3-(2-naphthyl)-D,L-alanyl, andN-Ac-3-(2,4,6-trimethylphenyl)-D,L-alanyl;B is an amino acyl residueselected from the group consisting of D-phenylalanyl,D-p-chlorophenylalanyl, D-p-fluorophenylalanyl, D-p-nitrophenylalanyl,2,2-diphenylglycyl, D-α-methyl-p-chlorophenylalanyl and3-(2-naphthyl)-D-alanyl; C is an amino acyl residue selected from thegroup consisting of D-tryptophanyl, D-phenylalanyl,3-(3-pyridyl)-D-alanyl, and 3-(2-naphthyl)-D-alanyl; D is an amino acylresidue selected from the group consisting of L-phenylalanyl, L-tyrosyl,and 3-(3-pyridyl)-alanyl, arginyl, or G; E is 3-(2-naphthyl)-D-alanyl,3-(3-pyridyl)-D-alanyl, D-tyrosyl, D-tryptophanyl, D-nicotinyl-lysyl,pyridylacetyl-lysyl, D-Glu(AA) or G; F is an amino acyl residue selectedfrom the group consisting of L-leucyl, L-norleucyl, L-phenylalanyl,L-tryptophanyl, and 3-(2-naphthyl)-L-alanyl; G is an amino acyl residueselected from the group consisting of the radicals represented by thefollowing structural formulas: ##STR7## wherein n is 1 to 5; R¹ is alkylof 1 to 6 carbon atoms or fluoroalkyl; R² is hydrogen or R¹ ; or R¹--HN--C═NR² is a ring represented by the following structure formulas:##STR8## wherein m is 1 to 4; A is hydrogen or alkyl of 1 to 6 carbonatoms; and X is halo or A; and ##STR9## wherein R³ is hydrogen, alkyl of1 to 6 carbon atoms, phenyl or phenylloweralkyl; and J is D-alaninamide;D-leucinamide; glycinamide; or --NHR⁴ wherein R⁴ is lower alkyl orNHCONH₂.
 2. A compound of claim 2 wherein:A is N-Ac-D-Nal(2) orN-Ac-D-pCl-Phe; B is D-pCl-Phe or D-pF-Phe; C is D-Trp, D-Nal(2) orD-Pal(3); D is Pal(3), Tyr, Arg, Deh, Mbh, Bth or Pha; E is D-Trp,D-Tyr, D-Nal(2), D-Pal(3), D-Deh, D-Bth, D-Mbh or D-Pha; F is Leu orPhe; G is Deh, Bth, Mbh, or Pha; and J is D-AlaNH₂ or GlyNH₂ ; or apharmaceutically acceptable salt thereof.
 3. A compound of claim 2wherein:A is N-Ac-D-Nal(2); B is D-pCl-Phe; C is D-Trp or D-Pal(3); D isTyr, Arg, Deh, Mbh, Bth or Pha; F is Leu; and J is D-AlaNH₂ ; or apharmaceutically acceptable salt thereof.
 4. A compound of claim 3wherein G is Deh or Bth or a pharmaceutically acceptable salt thereof.5. A compound of claim 4 wherein G is Bth or a pharmaceuticallyacceptable salt thereof.
 6. A compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-E-Leu-G-Pro-D-AlaNH₂ wherein E isD-Pal(3) or D-Trp; or a pharmaceutically acceptable salt thereof.
 7. Acompound of claim 6 wherein C and E are the same or a pharmaceuticallyacceptable salt thereof.
 8. A compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Tyr-E-Leu-G-Pro-D-AlaNH₂, wherein E isD-Deh, D-Bth, D-Mbh or D-Pha; or a pharmaceutically acceptable saltthereof.
 9. A compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-Arg-E-Leu-G-Pro-D-AlaNH₂, wherein E isD-Trp, D-Pal(3), D-Nal(2), or D-Tyr; or a pharmaceutically acceptablesalt thereof.
 10. A compound of claim 9 wherein E is D-Tyr;or apharmaceutically acceptable salt thereof.
 11. A compound of claim 9wherein E is D-Trp or D-Pal(3);or a pharmaceutically acceptable saltthereof.
 12. A compound of claim 11 wherein C and E are the same;or apharmaceutically acceptable salt thereof.
 13. A compound of claim 3which isN-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Leu-G-Pro-D-AlaNH₂, wherein Dis Deh, Bth, Mbh or Pha, and E is D-Tyr, D-Nal(2), D-Trp or D-Pal(3); ora pharmaceutically acceptable salt thereof.
 14. A compound of claim 13wherein E is D-Tyr or D-Pal(3) or a pharmaceutically acceptable saltthereof.
 15. A compound of claim 3 which isN-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-E-Leu-G-Pro-D-AlaNH₂, wherein E isD-Trp, D-Tyr, or D-Nal(2);or a pharmaceutically acceptable salt thereof.16. A compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-C-Ser-D-E-Leu-G-Pro-D-AlaNH₂, wherein D and Gare independently Deh, Bth, Mbh or Pha, and C and E are independentlyD-Trp or D-Pal(3); or a pharmaceutically acceptable salt thereof.
 17. Acompound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-G-Pro-D-AlaNH₂ ;or a pharmaceutically acceptable salt thereof.
 18. A compound of claim 3selected from the group consistingof:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCL-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ; andN-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ; or apharmaceutically acceptable salt thereof.
 19. A compound of claim 3selected from the group consistingof:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCL-Phe-D-Trp-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Mbh-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;and N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 20. A compound of claim3 selected from the group consistingof:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;and or a pharmaceutically acceptable salt thereof.
 21. A compound ofclaim 3 selected from the group consistingof:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Tyr-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Tyr-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Tyr-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Trp-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Deh-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pha-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Deh-D-Nal(2)-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Mbh-D-Nal(2)-Leu-Mbh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Bth-D-Nal(2)-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Pha-D-Nal(2)-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Deh-Pro-D-AlaNH.sub.2;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Pha-Pro-D-AlaNH.sub.2; andN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH.sub.2; or a pharmaceutically acceptable salt thereof.
 22. A compound of claim3 selected from the group consistingof:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂; N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Deh-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Phe-Leu-Pha-Pro-D-AlaNH₂ ;N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;and N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 23. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 24. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 25. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 26. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(2)-Ser-Tyr-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 27. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ; ora pharmaceutically acceptable salt thereof.
 28. A compound of claim 3whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Bth-Pro-D-AlaNH₂ ; ora pharmaceutically acceptable salt thereof.
 29. A compound of claim 3whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Arg-D-Trp-Leu-Pha-Pro-D-AlaNH₂ ; ora pharmaceutically acceptable salt thereof.
 30. A compound of claim 3whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Bth-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 31. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Pha-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 32. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 33. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Pal(3)-D-Pal(3)-Leu-Bth-Pro-D-AlaNH.sub.2; or a pharmaceutically acceptable salt thereof.
 34. A compound of claim3 whichis:N-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Arg-D-Pal(3)-Leu-Deh-Pro-D-AlaNH₂; or a pharmaceutically acceptable salt thereof.
 35. A pharmaceuticalcomposition for inhibiting ovulation in a mammalian female subject;preventing ovarian hyperstimulation in response to exogenousgonadotropins, treating premenstrual syndrome, or treating endometriosisin a female human subject; for treating prostatic hypertrophy orinhibiting spermatogenesis in a male mammalian subject; or for treatingprecocious puberty in a human subject; or interrupting heat in a femaleanimal; or terminating pregnancy in a female mammalian subject;comprising an effective amount of the compound of claim 1 in admixturewith at least one pharmaceutically acceptable excipient.
 36. A compoundof claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Bth-D-Tyr-Leu-Bth-Pro-D-Ala-NH₂ ;or an optical isomer thereof; or a pharmaceutically acceptable saltthereof.
 37. The compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ; oran optical isomer thereof; or a pharmaceutically acceptable saltthereof.
 38. The compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Trp-Ser-Tyr-D-Pha-Leu-Pha-Pro-D-AlaNH₂ or anoptical isomer thereof; or a pharmaceutically acceptable salt thereof.39. The compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Deh-Leu-Deh-Pro-D-AlaNH₂ ;or an optical isomer thereof; or a pharmaceutically acceptable saltthereof.
 40. The compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Tyr-D-Bth-Leu-Bth-Pro-D-AlaNH₂ ;or an optical isomer thereof; or a pharmaceutically acceptable saltthereof.
 41. The compound of claim 3 whichisN-Ac-D-Nal(2)-D-pCl-Phe-D-Pal(3)-Ser-Mbh-D-Pal(3)-Leu-Mbh-Pro-D-AlaNH₂; or an optical isomer thereof; or a pharmaceutically acceptable saltthereof.